1. Field of the Invention
The present invention relates to genes coding for phenylalanine dehydrogenase, plasmids containing the gene, microorganisms transformed with the plasmid, a process for the production of phenylalanine dehydrogenase, and a process for the production of L-phenylalanine using an enzyme.
2. Description of the Related Art
Attempts have been made to produce L-amino acids using .alpha.-ketocarboxylic acid as a substrate. For example, a process for preparing L-glutamic acid by adding .alpha.-ketoglutarate and various kinds of amino acids to microbial cells (Katagiri et. al. Amino Acid and Nucleic Acid, 2, 18, 1960); a process for obtaining L-phenylalanine by adding L-glutamic acid or L-aspartic acid to a reaction mixture containing phenylpyruvic acid (Asai et. al., Amino Acid and Nucleic Acid, 2, 114 (1960); and a process for synthesizing L-tryptophan by adding L-glutamic acid or L-aspartic acid to a reaction mixture containing indolepyruvic acid (Aida et. al., Journal of General and Applied Microbiology, 4, 200, 1958, have been disclosed.
Japanese Unexamined Patent Publication No. 60-164493 describes a process for the production of L-phenylalanine by either culturing one of various kinds of microorganisms with phenylpyruvic acid and an amino group donor, or incubating cells of that microorganism or a treated product of the cells with phenylpyruvic acid and an amino group donor. However, this specification does not disclose just what kind of enzymes participate in the reaction. Moreover, such processes employ amino acids, which are an expensive amino group donor.
All of the above-mentioned processes use, as an amino group donor of an aimed amino acid, another amino acid, and are fundamentally different from a process of the present invention which uses an ammonium ion as an amino group donor, which is not so expensive. Namely, the prior art processes are more expensive than the present process. Moreover, the enzymes involved in the prior art process are different from those of the present process.
Japanese Unexamined Patent Publication No. 60-43391 discloses a process for production of L-amino acid wherein a microorganism capable of converting an .alpha.-keto acid to a corresponding L-amino acid is cultured, and during the culturing, the .alpha.-keto acid is fed into the culturing medium to convert the .alpha.-keto acid to the L-amino acid. According to the reaction mechanism suggested in the specification, as an amino group donor for the formation of an aimed L-amino acid from a corresponding .alpha.-keto acid, L-glutamate is used, which means that the reaction is carried out by an amino transferase. Moreover, the application discloses only Brevibacterium, Corynebacterium, and Escherichia coli as microorganisms involved.
Japanese Unexamined Patent Publication No. 59-198972 describes phenylalanine dehydrogenase and a process for the production of L-.alpha.-amino carboxylic acid using that enzyme. However, the phenylalanine dehydrogenase described therein is derived from Brevibacterium, and the specification does not suggest that Sporosarcina and Bacillus produce a similar enzyme. Moreover, the disclosed phenylalanine dehydrogenase has a molecular weight of 130,000.+-.10,000 and consists of subunits having a molecular weight of 66,000.+-.5,000 and, therefore, is different from the present phenylalanine dehydrogenase.
Japanese Unexamined Patent Publication No. 60-160890 discloses a process for the production of L-phenylalanine by either culturing one of various kinds of microorganisms with phenylpyruvate in the presence of an energy source, an inorganic ammonium compound or urea, and oxygen, or by incubating a cultured product of the microorganism or treated product thereof with phenylpyruvate in the presence of an energy source, an inorganic ammonium compound or urea, and oxygen. However, the specification does not suggest the kind of enzymes involved in the process, and the process is supposed to be essentially a fermentation process, due to the necessity for the presence of oxygen. Moreover, the specification does not refer to Sporosarcina.
Japanese Unexamined Patent Publication No. 60-24192 discloses the production of L-phenylalanine using a Corynebacterium strain transformed with a plasmid containing genes related to L-phenylalanine production derived from Corynebacterium.
Japanese Unexamined Patent Publication No. 60-62992 discloses the cloning of genes related to the synthesis of L-phenylalanine in Escherichia coli, and the expression of that gene under the control of a trp promoter to produce L-phenylalanine.
Japanese Unexamined Patent Publication Nos. 60-66984 and 60-210993 disclose the synthesis of L-phenylalanine using Brevibacterium microorganism transformed with a plasmid incorporating a gene for an enzyme related to L-phenylalanine synthesis, which gene is from L-glutamate-producing Coryneform. phenylalanine dehydrogenase genes of Bacillus and Sporosarcina origin, plasmids containing that gene, microorganisms transformed with the plasmid, and a process for the production of L-phenylalanine have not been described.
Note, microorganism producing phenylalanine dehydrogenase, a process for production of phenylalanine dehydrogenase, phenylalanine dehydrogenase per se, and a process for production of .alpha.-amino acid including L-phenylalanine are described in detail in E.P.C publication No. 0 206 460.
Generally, the ability of wild microorganism strains to produce useful substances is low, and therefore, when the production of useful substances is intended, using a microorganism, an improvement of microorganism is attempted to increase the ability of the microorganism to produce a target substance. However, conventional processes for the improvement of a microorganism, such as artificial mutagenesis using, for example, a chemical mutagen or physical mutagen such as UV rays, are time-consuming and depend greatly on chance.